N-benzyl-N-methyldecan-1-amine, derived from garlic, and its derivative alleviate 2,4-dinitrochlorobenzene-induced atopic dermatitis-like skin lesions in mice

Given the intricate etiology and pathogenesis of atopic dermatitis (AD), the complete cure of AD remains challenging. This study aimed to investigate if topically applying N-benzyl-N-methyldecan-1-amine (BMDA), derived from garlic, and its derivative [decyl-(4-methoxy-benzyl)-methyl-1-amine] (DMMA) could effectively alleviate AD-like skin lesions in 2,4-dinitrochlorobenzene (DNCB)-treated mice. Administering these compounds to the irritated skin of DNCB-treated mice significantly reduced swelling, rash, and excoriation severity, alongside a corresponding decrease in inflamed epidermis and dermis. Moreover, they inhibited spleen and lymph node enlargement and showed fewer infiltrated mast cells in the epidermis and dermis through toluidine-blue staining. Additionally, they led to a lower IgE titer in mouse sera as determined by ELISA, compared to vehicle treatment. Analyzing skin tissue from the mice revealed decreased transcript levels of inflammatory cytokines (TNF-α, IL-1β, and IL-6), IL-4, iNOS, and COX-2, compared to control mice. Simultaneously, the compounds impeded the activation of inflammation-related signaling molecules such as JNK, p38 MAPK, and NF-κB in the mouse skin. In summary, these findings suggest that BMDA and DMMA hold the potential to be developed as a novel treatment for healing inflammatory AD.

BMDA and its derivative DMMA, synthesized using the reductive amination method 26 , were previously identified in our study 27 .DMMA was herein included to investigate the impact of structural variation on biological function, potentially suggesting a functional group for anti-inflammatory drug design.BMDA (0.3%) or DMMA (0.3%) was incorporated into a base cream containing corn oil, olive wax, and distilled water, as outlined in Table 1.AD-like symptoms were induced according to the scheme presented in Fig. 1A.After three consecutive treatments with 1% DNCB, 0.5% DNCB was applied to the dorsal skin three times per week for two weeks to induce AD-like symptoms.We observed that all Balb/C mice exhibited AD-like phenotypes, including hemorrhage, swelling, hives, and erosion (Fig. 1B,C).As a positive control, a steroid drug such as prednisolone valeroacetate (0.15%) was utilized.Due to the generally thinner and more sensitive nature of mouse skin compared to humans 28,29 , a steroid cream with mild potency was chosen.Notably, topical application of BMDA or DMMA on the mice's skin resulted in the recovery from hemorrhage, swelling, and erosion induced by DNCB treatment, as depicted in Fig. 1B,C.Throughout the time course for AD-like skin lesion induction, AD-like symptoms peaked seven days after the first DNCB treatment and remained consistent for two weeks (Fig. 1D).However, during BMDA or DMMA administration, AD-like phenotypes were significantly alleviated 14 days after the first DNCB treatment (Fig. 1D).Following hematoxylin-eosin staining and measurement of epidermis and dermis thickness in the mice, it was noted that BMDA or DMMA administration substantially reduced the increased thickness of epidermis and dermis caused by DNCB treatment (Fig. 2A,C).These findings indicate that BMDA and DMMA have a beneficial effect in addressing AD symptoms.To explore the involvement of lymphoid organs from DNCB-treated mice in the therapeutic effect of BMDA and DMMA, we assessed the sizes of the spleen and inguinal lymph nodes in vehicle-, BMDA-, DMMA-, and steroid-treated mice during DNCB treatment.As illustrated in Fig. 3A-C, both BMDA and DMMA application significantly reduced the size and weight of the spleen, along with the size of the inguinal lymph nodes, which had expanded during DNCB treatment (Fig. 3A-C).The DNCB-induced skin disease model emulates AD, but unlike AD, DNCB induces acute inflammation.Thus, to determine whether mast cells, pivotal in the early stage of AD 6,30 , are implicated in this DNCB-induced AD-like condition, we conducted toluidine-blue staining.The results demonstrated that the infiltration of mast cells into the epidermis and dermis of DNCB-treated mice was impeded by the administration of BMDA or DMMA, mirroring the effect observed with steroid treatment (Fig. 4A,B).Additionally, given that an elevation in IgE titer is a significant immunological biomarker for the pathogenic progression of AD, we quantified IgE titers across the groups using specific ELISA.Application of BMDA or DMMA on the skin of DNCB-treated mice yielded lower IgE titers in their sera compared to vehicle application (Fig. 4C).These findings suggest that BMDA and DMMA exhibit restraint of mast cell infiltration and IgE production in DNCB-induced dermatitis.

Administration of BMDA or DMMA decreases the expression of inflammatory cytokines, iNOS, and COX-2 transcripts in the skin of DNCB-treated mice
Given our observation of BMDA and DMMA's inhibition of immune cell activities, including mast cells and B cells (Fig. 4A-C), we directed our attention to inflammatory mediators such as cytokines, nitric oxide (NO), and prostaglandin E2 (PGE2) originating from keratinocytes and immune cells residing in skin tissues.After extracting total RNAs from the skin tissues of vehicle-, BMDA-, DMMA-, and steroid-treated mice during DNCB treatment, we quantitatively assessed the expression of inflammatory cytokines, IL-4 (type 2 immune response), iNOS, and COX2 transcripts via RT-PCR.As depicted in Fig, 5A-C, transcript levels of TNF-α, IL-1β, and IL-6 inflammatory cytokines were down-regulated by topical application of BMDA or DMMA, mirroring the effect observed with steroid treatment.In addition, the elevated expression levels of IL-4 transcript due to DNCB treatment were reduced by topical administration of BMDA or DMMA (Fig. 5D).Similarly, transcript levels of iNOS and COX2 exhibited the same trend (Fig. 5E,F).These findings suggest that BMDA and DMMA could inhibit the expression of inflammatory mediators at the transcriptional stage.

The regimen of BMDA or DMMA diminishes the activation of MAPK and NF-κB singling pathways related to inflammation in the skin of DNCB-treated mice
We delved deeper into whether signaling pathways linked to the production of inflammatory mediators were influenced by the BMDA or DMMA regimen.As demonstrated in Fig. 6A,B and iNOS, we discovered that phosphorylation levels of NF-κB were significantly inhibited through BMDA or DMMA topical application, akin to the effects observed with steroid treatment (Fig. 6A,B).These findings indicate that both BMDA and DMMA exhibit anti-inflammatory properties by deactivating JNK and p38 MAPK, along with down-regulating NF-κB.

Discussion
For the first time, we present that the topical application of BMDA and DMMA on skin tissues elicits antiinflammatory effects on DNCB-induced AD-like symptoms.These effects can be attributed to the inactivation of p38MAPK-JNK signaling and the down-regulation of the NF-κB transcription factor.Consequently, BMDA or DMMA treatment curtailed the production of inflammatory cytokines like TNF-α, IL-1β, IL-6, IL-4 featuring a type 2 immune response, and inflammatory mediator proteins including iNOS and COX2.Based on our findings, we propose a diagram to depict anti-inflammatory effects of BMDA or DMMA on DNCB-induced AD-like symptoms (Fig. 7).Numerous reports have extensively documented the pivotal roles of mast cells in AD 6,31,32 .Based on several lines of evidence, we speculate that repeated exposure to DNCB triggers B cells expressing IgE, resulting in IgE secretion.This IgE then binds to Fcε receptors on mast cells, prompting the release of secretory granules containing histamine and PDE2, alongside inflammatory cytokines like TNF-α, IL-1β, and IL-6.In this context, our study reveals that BMDA and DMMA effectively inhibit the infiltration of mast cells into the epidermis and dermis, as demonstrated by toluidine-blue staining.This could elucidate how these small compounds impede the inflammatory cascade reaction.
Moreover, numerous studies have documented inflammatory cytokines such as TNF-α, IL-1β and IL-6 activate JNK and p38 MAPK leading to the translocation of NF-κB to the nucleus 33,34 .In the canonical NF-κB pathway, a dimer complex composed of RelA (p65) and p50 translocates into the nucleus 35,36 .During this process, NF-κB (RelA; p65) undergoes phosphorylation at the Ser563 residue [37][38][39] .Given the observed upregulation of TNF-α and IL-1β expression under DNCB-induced inflammation, we assumed activation of the canonical signaling of NF-κB based on the literatures.Consequently, we noted that BMDA and DMMA reduced phosphorylation of NF-κB (RelA; p65) at Ser536 residue.These molecules also decreased the phosphorylation levels of JNK and p38MAPK.However, these results do not necessarily imply a direct targeting of JNK, p38 MAPK, and NF-κB proteins by BMDA and DMMA.Instead, we propose that BMDA and DMMA may indirectly be associated with a decrease in typical TNF-α/IL-1β-mediated inflammatory signaling.Moreover, our previous study demonstrated that BMDA and DMMA inhibited LPS-induced signaling, while enhancing NRF2 and HO-1 expression 27 .We propose that these small molecules might be involved in blocking other inflammatory signaling pathways such as TLR4-MyD88-TRAF6 signaling or NRF2-HO1 signaling.Currently, we are actively under investigation to explore target proteins for BMDA and DMMA using click chemistry techniques 40,41 .
Interestingly, upon careful comparison of the anti-inflammatory efficacy between BMDA and DMMA, DMMA displayed a slightly superior anti-inflammatory effect on DNCB-induced AD-like phenotypes than BMDA.These distinct outcomes could potentially be linked to DMMA's structural characteristics.Specifically, the inclusion of a methoxy (-OCH3) group on the benzene ring of BMDA in DMMA's chemical structure might contribute to its water solubility (hydrophilicity).Supplementary Fig. 1 illustrates that benzyl-dodecyl-methylamine (referred to as FMJ-G2) encompasses dodecane, differing by 2 additional CH2 groups compared to BMDA.This implies that FMJ-G2 could be more hydrophobic than BMDA, impacting its solubility and potentially its interaction with target molecules within cells.Notably, the application of FMJ-G2 did not mitigate erythema, erosion, and excoriation; furthermore, it resulted in relatively larger spleen and inguinal lymph node sizes compared to other derivatives (Supplementary Fig. 1).On the other hand, decyl-(4-fluoro-benzyl)-methyl-amine (termed as FMJ-G3), which features the attachment of a fluoride (-F) group to the benzene ring, demonstrated significant anti-inflammatory effects on DNCB-induced skin lesions.These findings strongly indicate that the observed differential anti-inflammatory efficacy may arise from variations in the chemical structures between BMDA and its derivatives.In our forthcoming study, we plan to further explore the validity of this hypothesis by conducting pharmacokinetic analysis on these derivatives.

Animal
Balb/C mice (22-25 g, 6 weeks old) were procured from Orient Bio Inc. (Seongnam, Korea).The mice were divided into five groups: untreated, BMDA, DMMA, steroid and vehicle (a base cream).Each group consisted of five animals and none died during the animal experiment.The mice were kept under specific pathogen-free conditions with unrestricted access to diet and tap water.The housing conditions included a 12-h light/dark cycle at 22 °C and 50-55% humidity.

Figure 1 .
Figure 1.Topical application of BMDA or DMMA reduces the severity of AD-like skin lesion in DNCB-treated mice (A) The schedule of BMDA or DMMA treatment in DNCB-treated mice (B-D) Photographs of skin lesions from DNCB-induced Balb/C mice (n = 5 mice per group) treated with BMDA (0.3%), DMMA (0.3%), steroid (0.15%) or vehicle (a base cream) for 14 days.Representative images are shown.Skin thickness from untreated mice was measured using a caliper and served as a basis for comparison with other mouse groups.(***p < 0.001, against vehicle).

Figure 2 .
Figure 2. Effect of BMDA or DMMA treatment on epidermis and dermis thickness in DNCB-induced AD skin lesions (A-C) Skin lesions from DNCB-treated Balb/C mice, applied with BMDA, DMMA, steroid or vehicle for 14 days, were stained with hematoxylin-eosin solution after fixation with 10% formalin and paraffin section.The thickness of the epidermis and dermis from the mice were measured after hematoxylin-eosin staining.(**p < 0.01, ***p < 0.001, against vehicle).

Figure 3 .
Figure 3.Effect of BMDA or DMMA administration on lymphoid organs in DNCB-treated mice (A-C) Spleen and inguinal lymph node sizes and weights were measured in DNCB-treated Balb/C mice, applied with BMDA, DMMA, steroid, or vehicle for 14 days.The spleen and inguinal lymph nodes were demounted for the measurements.(*p < 0.05, ***p < 0.001, against vehicle).

Figure 4 .
Figure 4. Effect of BMDA or DMMA application on allergic reaction in DNCB-treated mice (A-D) Skin lesions from DNCB-treated Balb/C mice, applied with BMDA, DMMA, steroid, or vehicle for 14 days, were stained with toluidine-blue solution after fixation with 10% formalin and paraffin section.The number of infiltrating mast cells into the epidermis and dermis was counted from five randomly selected fields under microscopy.The scale bars indicate 100 µm.Additionally, blood samples were taken from the DNCB-treated Balb/C mice, applied with BMDA, DMMA, steroid, or vehicle for 14 days, and IgE titers in the sera were thereafter measured using its specific ELISA.(***p < 0.001, against vehicle).

Figure 5 .
Figure 5.Effect of BMDA or DMMA regimen on transcript levels of inflammatory cytokines and mediator proteins in the skin from DNCB-treated mice (A-F) Transcript levels of inflammatory cytokines (TNF-1α, IL-1β and IL-6), IL-4, and mediator proteins (iNOS and COX2) were measured by qRT-PCR from the skin tissues of the mice.(***p < 0.001, against vehicle).

Figure 6 .
Figure 6.Effect of BMDA or DMMA treatment on signaling molecules involved in inflammation in the skin from DNCB-treated mice (A, B) Protein lysates from the skin tissues of DNCB-treated Balb/C mice, applied with BMDA DMMA, steroid, or vehicle for 14 days, were prepared.The phosphorylation levels of signaling molecules involved in inflammation were analyzed with immunoblotting using their specific antibodies.Phosphorylation sites of JNK indicate Thr183 and Tyr185 residues in JNK.Phosphorylation sites of p38MAPK denote Thr180 and Tyr182 residues in p38MAPK.Phosphorylation site of NF-κB (p65; RelA) designates Ser536 residue in NF-κB.The relative expression of the proteins was analyzed after scanning using AlphaView, version 3.2.2(Cell Biosciences Inc., Santa Clara, CA, USA).(*p < 0.05, **p < 0.01 and ***p < 0.001, against vehicle).

Figure 7 .
Figure 7. Efficacy of BMDA or DMMA on DNCB-induced inflammation in the skin of mice DNCB sensitization on the skin induces the activation of JNK, p38 MAPK, and NF-κB, resulting in an increase in the expression of inflammatory cytokines (TNF-α, IL-1β, and IL-6) and mediator proteins (iNOS and COX2).However, treatment with BMDA or DMMA inhibits the phosphorylation of JNK, p38 MAPK, and NF-κB, leading to a downregulation in the transcription of inflammatory cytokines (TNF-α, IL-1β, and IL-6), IL-4, and mediator proteins (iNOS and COX2).Consequently, this diminishes allergic reaction such as an increase of the infiltration of mast cells and IgE titer, ultimately alleviating the severity of swelling, rash, and excoriation induced by DNCB in the skin.

Table 1 .
Composition of cream (10 mL) for topical application on the mouse skin.